Abstract Different distributions of ridged and smooth morphologies of Nucella emarginata are found at wave-exposed and protected sites at China Point in Monterey Bay. A greater number of the ridged morphology was found at the protected site and the smooth at the exposed. Variation in drag coefficients, adhesive force, and crushing force between shell morphologies do not appear to dictate this difference in distribution, although adhesive force is marginally greater in wave exposed whelks. Predation on tethered whelks is greater in the ridged morphology in general than the smooth. However, whelks were eaten more quickly at the protected site, suggesting that wave action's effect on predators may play a role in the distribution of the two morphologies. In a laboratory experiment, crabs showed no preference between ridged or smooth N. emarginata, with a slight preference for exposed whelks. Äfter 27 days of growth, it was unclear whether differences in shell morphology were present among juveniles hatched from the exposed site. Introduction An important issue in ecology is understanding how different genetic and environmental factors dictate differences in the morphology of individuals within a species. In turn it is important to see whether or not different morphologies lead to greater or decreased individual fitness. Natural selection may act on genetically based morphological variation to produce evolutionary change. Environmentally induced phenotypic changes may have important effects at the population level. Shell morphology is used as a rather broad term in the literature referring to everything from architecture of the actual shell to differences in aperture width and length. Morphological differences in the shell are often described as shell sculpture. Differences in shell sculpture have been shown to protect against certain predators in marine snails (e.g. Vermeij 1993). Differences in shell sculpture in bivalves has been show to reduce drag in burrowing (e.g. Vermeij 1993). Clearly, morphological (and specific sculptural) differences have the potential to affect fitness. Nucella emarginata is a common predatory whelk found in rocky intertidal areas, along the California coast. This species has direct development, juvenile snails hatch from egg capsules attached to rock surfaces in the intertidal zone. Shell sculpture in N. emarginata has been found to be under genetic control (e.g, Palmer 1984). However, differences in shell morphology, specifically aperture thickness, have been found within populations of N. emarginata, aperture thickness being a defense against crab predation (Geller 1990). Whereas morphology can be genetic, crab presence has also been shown to produce morphological changes in intertidal snails (e.g. Trussell 1996). Thus, while shell sculpture can be a genetic trait, the possibility of predator induced phenotypic plasticity remains to be answered for N. emarginata. At China Point in Monterey Bay, California, two shell morphologies of Nucella emarginata exist. A ridged morphology has raised axial ridges on the shell, while a smooth morphology lacks these ridges and has a smooth outer shell. Preliminary observations suggested that these two morphologies were unevenly distributed between wave exposed and wave protected sites at China Point. More ridged snails were observed in wave protected sites, and more smooth snails were observed in wave exposed sites. In this study, I examined several factors that could be influencing the different distributions of N. emarginata morphologies. Wave impact could be dictating these different distributions by ripping off one morphology with greater drag. Wave impact could also be affecting the predators of the snails. The main predators of N. emarginata at China point are the crab Cancer antennarius and the Pisaster ochraceus, particularly at the wave exposed site. Power supplied by breaking waves has been shown to protect intertidal residents by knocking away their enemies or preventing them from feeding (Leigh et al. 1987). Crab predation at the exposed site could be similarly affected because in more wave exposed sites crab have a harder time locomoting and therefore feeding. Differences in the crushing forces tolerated by ridged and smooth snails could be dictating a preference in crab predation and therefore affecting the distributions seen at each site. Initial distributions at hatching must also be determined before we can accurately gauge the affect of either wave impact or predation. In this study the most significant factor found to affect the difference in distribution was wave impact on predators. Whether distributions were the same at hatching as at later in life was indeterminate in the time allotted for the study. Methods & Materials Morphology Two distinct shell morphologies of Nucella emarginata were examined: smooth and ridged. Ridged morphologies have raised axial ridges of varying degrees. Smooth morphologies have smooth shells lacking any such ridges. While some intermediate morphologies were found, I focused my study on N. emarginata with distinct morphologies. N. emarginata's shell morphology was compared in sites with differing wave impact. A wave-exposed site was selected in Monterey Bay at the western edge of China Point, and a protected site at Fisher beach, near the Monterey Bay Aquarium. I quantified the difference in shell morphology at these sites by choosing at each site a 2 m by 1 m rock area with a great concentration of N. emarginata, and counting the number of each shell morphology present in that area. Drag To assess the potential impact of waves, the drag coefficient was measured for each morphology. To this end, I collected five ridged-morphology N. emarginata from the protected site and five smooth from the exposed site. These whelks were boiled and their soft bodies removed. Each snail was then glued with five-minute epoxy to the plastic plate of a drag transducer and placed in a high-speed wind tunnel. The plate of the transducer was held flush with the wall of the tunnel to simulate the orientation of the snail in surf-zone flow. The drag imposed by wind was transduced to a voltage and recorded using a voltmeter. The direction of greatest drag was determined, and force readings were taken at five different wind speeds: 21.4 m/s, 28.9 m/s, 36.4 m/s, 43.9 m/s and 51.3 m/s. Profile area for each snail was determined by taking a digital picture of the snail next to an object of a known surface area. The object and the snail were cut out of the paper print-out and weighed. Profile area was determined using the ratio of paper weight to area. With this information in hand, the drag coefficient could be determined for each shell. Drag Coefficient = (2 *Drag Force) (Air density* Profile Area* (Velocity/2)) A two-tailed t test at a wind speed of 36.4 was used to determine whether a significant difference existed between the average drag coefficients of the two morphologies. Adhesive Force The adhesive force of N. emarginata was compared between the exposed and the protected sites by pulling off approximately 40 whelks at each site. Whelks were pulled off in the direction of greatest drag. Two different force meters were used to record adhesive force, one with a 500 g rating for whelks below 14 mm long, the other with a 5 kg rating for bigger whelks. A string was attached to the force meter with a loop and the other end was secured with a noose around the whelk's shell. The meter was then used to tug on the snail, and the dislodgment force was recorded. For whelks that were too small, or oriented in a position making it difficult to use the noose technique, the string was glued to the whelk's shell using cyanoacrylate adhesive. N. emarginata that were accessible and feeding on acorn barnacles were selected for these tests because whelks tend to hold on with greatest force while feeding. Crushing Force The force to crush shells (a simulation of predation by crabs) was measured on twenty whelks, ten from each site and five of each morphology. Crushing was standardized to larger crab predation, which involves crushing of the shell with one claw, as opposed to smaller crabs that peel away at the aperture lip of the snail (Bertness and Cunningham 1981). One jaw of a pair of pliers was held steady by a vise and the snail was placed between this stationary jaw and the movable jaw. A 5 kg force meter was then used to forcibly close the pliers until the aperture was crushed, and the crushing force was recorded. Each whelk was grasped in the same orientation and the meter was placed at approximately the same spot to avoid complications due to a variable lever arm. Tethering To examine predation at each site, twenty whelks, ten of each morphology, were transplanted from protected to exposed sites and vice versa. Fishing snap swivels were glued with epoxy to the whelk's shells and were attached with fishing line to bolts in the lower intertidal zone, just below the whelk's normal vertical distribution. Two snails of each morphology were tethered to each bolt at approximately the same level in the intertidal zone. Snails were checked 1,3 and 6 days after tethering. Crab Predation and Influence To analyze predation in the lab as well as to test for an effect of crab presence on shell growth, three crabs (Cancer antennarius approx. carapace width 5-8 cm) were collected and placed in Tupperware*“ containers (25*25 cm) with mesh cutouts to allow water flow. These Tupperware"M containers were then weighted with rocks and placed into a larger dish tub with water flowing in. Three control tubs were also set up in random order along the line of water flow. Eight whelks of similar length were placed in each of the crab and no-crab containers, four whelks from each site (two of each morphology), marked for their site of origin. Influence of crab effluent on whelks was determined in smaller 1-liter containers (Figure 1). Water was siphoned into these containers with plastic tubing from the larger tubs containing either a crab or no crab. Two containers with whelks from protected and exposed sites were set up for each larger tub. Four whelks of similar size (shell lengths - approx. 16 mm- 18 mm) were placed in each smaller container and individually labeled with nail polish. Initial and final readings were taken of length and width of the aperture at approximately the same position on the shell. Two notches were filed into the outer lip of the body whorl of each whelk to determine whether growth had occurred. Whelks were fed bay mussels (Mytilus trossulus) of approximately the same (shell length = approx. 20 mm to 22 mm). Hatching To test for differences in initial shell morphology distribution, I collected six sets of egg cases from four different egg clusters from both the exposed and the protected site and kept them in tea strainers. Only the egg cases from the exposed site hatched so 20 whelks from each of the four exposed containers were transferred to four new tea strainers and placed in containers with flowing seawater. The baby whelks were fed the smallest available mussel recruits (M. trossulus); mussels were increased in size as the snails grew. Snails maintained in this fashion were held for 27 days, at which time their morphology was assessed. Results Morphology There was a larger fraction of ridged shells at the protected site than at the exposed site (Figure 2). Drag The drag coefficients for smooth and ridged snails were not statistically different (two-tailed test, t=7.9, p» 0.05). At a wind speed of 36.4 m/s (equivalent to a water velocity of 3.2 m/s), the average drag coefficient for smooth snails was 0.630 with a standard error of 0.009. The average drag for ridged was 0.648 with a standard error of 0.019. Adhesive Force The difference in adhesive force between ridged and smooth snails was not significantly different. Statistical significance was determined by conducting an ANCOVA with site as the variant and whelk shell length as a covariant. Although there was no difference between morphologies (ANCOVA, F= 0.218, p =.642), a significant difference in adhesive force was found between snails at the two sites (ANCOVA, F = 7.698, p = 0.007) (Figure 3). The protected snails averaged 3.30 N while the exposed snails averaged 4.71 N. Crushing Force No significant difference in crushing force was found between the two morphologies, or between the two sites (ANCOVA, F = 1.013, p = 0.330). Average crushing force for exposed snails was 23.72 N (Standard Error, SE-4.43). Average crushing force for protected snails was 21.22 N (SE- 2.01). For ridged shells, the average crushing force was 22.94 N (SE-3.54), and average crushing force for smooth shells was 22.23 N (SE-3.60). Tethering A greater number of ridged snails were eaten at both the protected and the exposed sites, as seen in Figure 4. Over the 6 days of the experiment wave intensity varied, with larger waves on days 1-3 and low wave intensity on days 4-6. Exposed whelks were eaten at a greater rate during calmer wave activity, while protected snails were eaten at a relatively steady rate. Two predators, crabs and Pisaster, fed on exposed snails, while the protected snails had evidence only of crab predation. Fragments of shell attached to line suggested crab predation, whereas empty shells attached to line suggested Pisaster. Crab Predation In the laboratory experiment, crabs showed no preference for either ridged or smooth snails. Crabs chose to eat exposed snails first 4 out of 6 times (p = 0.23), but showed no further consistent preference for exposed or protected snails. No increase in the thickness of the aperture lip was seen in either crab-exposed snails or control snails. A significant difference (ANOVA, F=15.14, p-0.008) was seen in drilling rates between control snails and those under crab influence with crab f3 excluded (Table 1). This crab was unable to feed on whelks because it had lost half a claw. Whelks exposed to water flow from a tank of actively feeding crabs ate the least amount of mussels; twelve of these whelks didn’t feed at all (Figure 5). Hatching In the 27 days of this experiment, the hatched N. emarginata had not grown enough to distinguish between ridged or smooth morphologies. The whelks grew from 1 mm at time of hatching to 3 mm after 27 days. They also developed banding of coloration, and some initial signs of ridges or architecture, however if was impossible to discern different morphologies because all appeared the same. Discussion Wave impact on different shell morphologies of Nucella emarginata does not seem to dictate their ability to live in wave exposed or wave protected conditions. The drag coefficient is not significantly different, so the drag on the two different morphologies of the same size snail is approximately the same. Therefore, the greater abundance of smooth snails at the wave exposed site cannot be explained by lessened drag on the smooth morphology. The adhesive force between the two morphologies does not differ as would be expected if this force were a selective factor. However, the average adhesive force of the wave exposed N. emarginata gives them a greater ability to adhere in conditions of greater wave impact. This difference could be related to differences in foot size (Trussell 1997). Although wave impact on the snail itself cannot explain the different distributions of morphologies, its effect on predation appears to be a viable explanation. The crushing force of shells did not vary between the two sites, nor did it vary between morphologies. Therefore, the ability of the crab to crush shells is unlikely to dictate a preference by this predator. In the laboratory the crabs did not significantly favor snails of a particular morphology or those from a particular site, suggesting that crabs at China Point would not eat more of one type of N. emarginata. However, the tethering experiment yielded different results. The tethering experiment was conducted over a period of 6 days, the first three of which were wavy, and the last three of which were extremely calm. During the first three days, only one snail was eaten at the exposed site while eight were eaten at the protected site. These wavy days may have made it difficult for crab predators to prey upon the tethered snails at the exposed site. In contrast, on the calm days 8 more snails were eaten at the exposed site, while feeding rates stayed approximately the same at the protected site. The calm conditions may have allowed crab predators greater access to snail prey at the exposed site. Bigger waves and greater wave impacts at the exposed site limit crab mobility in the intertidal zone (Leigh et al. 1987). This suggests that the intensity of crab predation may be dictated by wave conditions. The contradictory results to the above experiments lay in the preference for ridged snails at the exposed site; many more ridged snails than smooth snails were eaten. At the exposed site eight out of the nine snails eaten were ridged. This could be due to unforeseen differences between predation in a laboratory experiment as opposed to predation in the field. Ridged snails may be easier for crabs to grasp than smooth snails. This could explain the greater abundance of smooth snails at the exposed site. However, a preference for ridged snails was also observed at the protected site, leaving uncertainties as to the factors dictating distributions of different morphologies there. While it is therefore probable that crab predation plays a key role in the different distribution of ridged and smooth N. emarginata, this factor cannot explain all distribution results. Plastic response to crab presence was indeterminate in these experiments due to the lack of feeding by many of the snails under crab influence. No similar trend was seen in the control snails, suggesting that crab presence causes decreased feeding rate in snails. The snails in the presence of the crab with half a claw had similar feeding rates to control snails, suggesting that crabs must be actively feeding on snails to have an affect. This experiment would have to be run for a longer period to see if snails add thicker shell in response to crabs despite lack of feeding. Crab effluent has been shown to cause snails to grow thicker shells in Littorina obtusa (Trussell 1996). The next step is to determine, by hatching and raising N. emarginata, if the underlying assumption of equal distribution for each population holds. If distributions at hatching are determined, crab predation could be shown to play a significant role. Results in the field suggest that there may be an advantage to having a smooth shell. It this is under genetic control, natural selection may favor smooth shells, and they could become more prevalent unless there are other advantages to having a ridged shell. Conclusion The direct impact of environment on the snail is not a major factor in the distribution of their shell morphology. In contrast, while different distributions of ridged and smooth Nucella emarginata were not explained completely, wave impact on predators of these snails seems to be very plausible explanation. To further explain why different morphologies of N. emarginata were seen in different numbers at these two sites, initial distributions would have to be determined. That information could clarify some of the inconsistencies in this experiment as well as the literature as to whether shell sculpture is exclusively genetic, or also a plastic trait. Acknowledgments This project owes a lot to the help of my advisors Mark Denny, Eric Sanford and Jim Watanabe. I would like to thank Mark Denny for always listening to and answering my numerous questions, as well as his creative ideas for how I was actually going to test all my hypotheses. Thanks to Eric Sanford for all his time and help with my experimental set-ups and ideas, from catching crabs to interpreting results. Thanks to Jim Watanabe for all his statistics help, as well as thoughtful suggestions along the way. I would also like to thank Carrie Kappel for always volunteering her time. I would also like to thank my Dad for helping me collect over 200 snails on his vacation. Literature Cited Bertness, M.D. and C. Cunningham. 1981. Crab shell crushing predation and gastropod architectural defenses. Journal of Experimental Marine Biology and Ecology Vol. 50 no. 2-3: 213-230. Geller, Jonathon B. 1990. Consequences of a morphological defense: growth, repair and reproduction by thin and thick-shelled morphs of Nucella emarginata. Journal of Experimental Marine Biology and Ecology. Vol.144, no.2,3: 173-184. Leigh Jr., Egbert G, Robert T. Paine, James F. Quinn and Thomas H. Suchanek. 1987. Wave energy and intertidal productivity. National Academy of Sciences Proceedings Vol. 84, no. 5: 1314-1318. Palmer, Richard A. 1984. Species Cohesiveness and genetic control of shell color and form in Thais emarginata. Malacologia Vol 25, no 2: 477-491. Trussell, Geoffrey C. 1996. Phenotypic plasticity in an intertidal snail: The role of the common crab predator. Evolution Vol 50, no 1: 448-454. Trussell, Geoffrey. 1997. Phenotypic plasticity in the foot size of an intertidal snail. Ecology Vol 78, no. 4: 1033-1048. Vermeij, Geerat J. 1993. A Natural History of Shells. New Jersey: Princeton University Press. Table 1:Analysis of Variance for differences in drilling rate between whelks of different sites and either under the influence of crabs or controls. Dep Var:Drilling n:10 Multiple R: 0.87 Source Sum of Squares Mean-Square F-ratio 15.14 Treat 98.82 98.82 Whelks 0.42 0.06 0.42 Treat Whelks 20.42 20.42 0.008 0.81 0.13 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure Legends Experimental set- up for crab predation and crab influence on snails. Predator preference for snail morphologies was assessed in the large containers, whereas effects of crabs on whelks was quantified in smaller containers with water flowing from the tubs. See text for details. Distribution of ridged and smooth morphologies of Nucella emarginata in samples taken at the wave exposed (n-62) and wave protected (n-62) areas. Black represents the ridged morphology while striped represents the smooth. The difference in the average adhesion between snails from the wave exposed and the wave protected areas. Exposed whelks had a greater adhesion force on average. Predation on tethered Nucella after-1, 3 and 6 days attached in the low intertidal zone. Days 1-3 had wavy conditions and days 4-6 were calm. Black represents snails transplanted from the wave exposed area to the waye protected area while striped represents snails transplanted from the waye protected area to the wave exposed area. Differences in drilling rates between snails under crab influence and control snails. Black represents snails from the wave exposed site and stripes represent snails from the wave protected site. Crab-exposed snails fed significantly less than control snails. O C O O 0 DE Z OE X 4 DE ZOE — Z OE 2 a a a a 2 7 V. LL 0 8 0 uee eonN 10 — L 9 V 8 o (N)o AIseupy *•. 8 0 o LL buissiui sijeus jo 4 0 10 O pelllip sjessnu jo Z